Systemic Carnitine Deficiency Gene and Uses Thereof

ABSTRACT

The gene responsible for systemic carnitine deficiency was found to be the OCTN2 gene involved in the transportation of organic cations. This invention enables tests for this disease by detecting whether or not the OCTN2 gene has a mutation. Furthermore, systemic carnitine deficiency can be treated using the normal OCTN2 gene and its protein.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/940,500, filed on Sep. 13, 2004, to issue as U.S. Pat. No. 7,413,860on Aug. 19, 2008, which is a divisional of U.S. application Ser. No.09/798,743, filed on Mar. 2, 2001, now U.S. Pat. No. 6,790,831, which isa continuation of PCT/JP99/04853, filed on Sep. 7, 1999, and claimspriority from Japanese Patent Application No. 10/252,683, filed on Sep.7, 1998. The contents of each of these applications are incorporated intheir entirety by reference herein.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING FILED ELECTRONICALLY

An electronic version of the Sequence Listing is filed herewith, thecontents of which are incorporated by reference in their entirety. Thecomputer-readable file, created on Aug. 15, 2008, is 64.0 kilobytes insize and titled 14875073003SeqList.txt.

1. Technical Field

This invention relates to molecules used in the testing and treatment ofsystemic carnitine deficiency, as well as methods for testing thedisease.

2. Background of the Invention

Systemic Carnitine Deficiency (SCD) is a human genetic disease inheritedthrough autosomal recessive inheritance, the main symptoms beingskeletal or cardiac muscle disorders (NIM 212140) (Roe, C. R. andCoates, P. M., Mitochondrial fatty acid oxidation disorder, Themetabolic and molecular bases of inherited diseases 7th ed., edited byScriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D., McGraw-Hill,New York, 1995, 1508-1509; Karpati, G. et al., The syndrome of systemiccarnitine deficiency: clinical, morphologic, biochemical, andpathophysiologic features, Neurology 1975, 25:16-24). Serum carnitinelevels and intra-tissue carnitine levels are known to be extremely lowin these patients compared to healthy individuals. Carnitine is anindispensable co-factor in the long-chain fatty acid metabolism. Acarnitine-mediated mechanism enables intracellular fatty acids topermeate mitochondrial outer and inner membranes, and energy is producedwhen these fatty acids undergo β-oxidation within the mitochondria(Walter, J. H., L-Carnitine, Arch Dis Child, 1996, 74:475-478; Bremer,J., Carnitine metabolism and functions, Physiol Rev, 1983, 1420-1480).The abnormal decrease of carnitine concentration in systemic carnitinedeficiency patients is thought to be the direct cause of diseases intissues such as muscles that require a large amount of energy. Membranephysiological studies done using fibroblasts from systemic carnitinedeficiency patients have shown that these cells lack the mechanism totransport carnitine from the outside of the cell to the inside. A genethat encodes a protein involved in this mechanism is presumed to be thegene responsible for this disease (Tein, I. et al., Impaired skinfibroblast carnitine uptake in primary systemic carnitine deficiencymanifested by childhood carnitine-responsive cardiomyopathy, PediatrRes, 1990, 28:247-255). However, the gene responsible for systemiccarnitine deficiency is yet to be isolated.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide the gene responsiblefor systemic carnitine deficiency. Moreover, this invention aims toprovide a molecule used in the testing and treatment of systemiccarnitine deficiency, as well as a method for testing the disease.

The Inventors isolated several genes encoding proteins involved in thetransport of organic cations. Among these, the Inventors discovered thehuman gene (human OCTN2 gene) having an activity to transport carnitinein a sodium ion dependent manner, and the corresponding mouse gene(mouse OCTN2 gene) (Japanese Patent Application Hei 9-260972, JapanesePatent Application Hei 10-156660). The Inventors thought that theisolated OCTN2 gene might be the gene responsible for systemic carnitinedeficiency, and evaluated this possibility.

Specifically, the nucleotide sequence of the OCTN2 gene of the systemiccarnitine deficiency mouse model and systemic carnitine deficiencypatients were analyzed. As a result, the Inventors discovered thepresence of various mutations in the OCTN2 gene of both the mouse modeland systemic carnitine deficiency patients. In other words, for thefirst time in the world, the Inventors succeeded in revealing thatsystemic carnitine deficiency is caused by mutations in the OCTN2 gene.

Moreover, due to the close relationship of OCTN2 gene mutation andsystemic carnitine deficiency, the Inventors found that this disease canbe tested by examining whether or not there is a mutation in the OCTN2gene of a patient.

It was also found that systemic carnitine deficiency could be treated byusing the normal OCTN2 gene and its protein, to complete the invention.

Therefore, this invention relates to molecules used in the testing andtreatment of systemic carnitine deficiency, as well as methods fortesting the disease. More specifically, the present invention relatesto:

(1) a DNA for testing systemic carnitine deficiency, wherein the DNAhybridizes to a DNA comprising the nucleotide sequence of SEQ ID NO:5,or the transcription regulatory region thereof, and comprises at least15 nucleotides;

(2) a molecule as in any one of (a) to (c) below, which is used for thetreatment of systemic carnitine deficiency,

(a) a protein comprising the amino acid sequence of SEQ ID NO: 1,

(b) a compound that promotes the activity of the protein comprising theamino acid sequence of SEQ ID NO: 1, or,

(c) a DNA encoding the protein comprising the amino acid sequence of SEQID NO:1;

(3) a pharmaceutical composition for treating systemic carnitinedeficiency, comprising a molecule of (2) as the active ingredient;

(4) a pharmaceutical composition for treating systemic carnitinedeficiency, comprising an antibody binding to the protein comprising theamino acid sequence of SEQ ID NO: 1 as the active ingredient;

(5) a test method for systemic carnitine deficiency comprising thedetection of a mutation in the DNA encoding the protein comprising theamino acid sequence of SEQ ID NO: 1, or the transcription regulatoryregion of said DNA;

(6) the test method for systemic carnitine deficiency of (5) comprisingthe steps of,

(a) preparing a DNA sample from a patient,

(b) amplifying patient-derived DNA using the DNA of (1) as a primer,

(c) cleaving the amplified DNA,

(d) separating the DNA fragments by their size,

(e) hybridizing the DNA of (1) labeled by a detectable label as a probeto the DNA fragments separated, and,

(f) comparing the size of the DNA fragment detected with a control froma healthy individual,

(7) the test method for systemic carnitine deficiency of (5) comprisingthe steps of,

(a) preparing an RNA sample from a patient,

(b) separating the prepared RNA by size,

(c) hybridizing the DNA of (1) labeled by a detectable label as a probeto the RNA fragments separated, and,

(d) comparing the size of the RNA fragment detected with a control froma healthy individual,

(8) the test method for systemic carnitine deficiency of (5) comprisingthe steps of,

(a) preparing a DNA sample from a patient,

(b) amplifying patient-derived DNA using the DNA of (1) as a primer,

(c) dissociating the amplified DNA to single-stranded DNA,

(d) separating the dissociated single-stranded DNA on a non-denaturinggel, and,

(e) comparing the mobility of separated single stranded DNA on the gelwith a control from a healthy individual,

(9) the test method for systemic carnitine deficiency of (5) comprisingthe steps of,

(a) preparing a DNA sample from a patient,

(b) amplifying patient-derived DNA using the DNA of (1) as a primer,

(c) separating the amplified DNA on a gel in which the concentration ofthe DNA denaturant gradually increases, and,

(d) comparing the mobility of separated DNA on the gel with a controlfrom a healthy individual.

The present invention is based on the finding by the present inventorsthat systemic carnitine deficiency is caused by a mutation in the genenamed “OCTN2”. First and foremost, this invention relates to a moleculeused in the testing and treatment of systemic carnitine deficiency, aswell as a method for testing the disease.

In the present invention, the genomic DNA region (for example, SEQ IDNO:5) containing OCTN2, or an oligonucleotide (probe and primer) thathybridizes to the nucleotide sequence of the regulatory region(comprising the intron, promoter, and enhancer sequences as well) ofOCTN2 is used.

This oligonucleotide preferably hybridizes specifically to the genomicDNA region containing OCTN2, or the regulatory region of OCTN2. Herein,“hybridizes specifically” indicates that cross-hybridization does notsignificantly occur with DNA encoding other proteins, under normalhybridizing conditions, preferably under stringent conditions (forexample, the conditions in Sambrook et al., Molecular Cloning secondedition, Cold Spring Harbor Laboratory Press, New York, USA, 1989).

When using as a primer, the oligonucleotide is usually, 15 to 100 bp,preferably, 17 to 30 bp. The primer may be any, as long as it canamplify at least a part of the OCTN2 gene or the region regulating itsexpression. Such regions comprise, for example, the exon region ofOCTN2, the intron region, the promoter region, and enhancer region.

On the other hand, the oligonucleotide used as a probe usually comprisesat least 15 bp or more if it is a synthetic oligonucleotide. It is alsopossible to use a double stranded DNA obtained from a clone incorporatedinto a vector such as plasmid DNA. The probe may be any, as long as itspecifically hybridizes to at least a part of the OCTN2 gene or theregion regulating the expression of the gene. Regions to which the probehybridizes include, for example, the exon region, intron region,promoter region, and enhancer region of the OCTN2 gene. When using asthe probe, oligonucleotide or double stranded DNA is suitably labeled.Examples of labeling methods are, phosphorylating the 5′ end of theoligonucleotide by ³²p using T4 polynucleotide kinase, and incorporatinga substrate nucleotide labeled by an isotope such as ³²p, a florescentdye, or biotin, using the random hexamer oligonucleotide as a probe andusing DNA polymerase such as the Klenow enzyme (random primingtechnique).

In the present invention, “a test method for systemic carnitinedeficiency” includes not only a test for patients showing symptoms ofsystemic carnitine deficiency caused by a mutation of the OCTN2 gene,but also a test for detecting a mutation of the OCTN2 gene fordetermining whether or not the person tested is likely to developsystemic carnitine deficiency arising from a OCTN2 gene mutation. Inother words, the risk of developing systemic carnitine deficiency maygreatly increase in cases where one of the OCTN2 alleles develops amutation, even when no symptoms are visible on the outside. Therefore,tests for specifying patients (carriers) having a mutation in an OCTN2allele are also included in the invention.

In the present invention, a test method for systemic carnitinedeficiency using the above oligonucleotides comprises the detection of amutation in the OCTN2 gene or its transcription regulatory region. Oneembodiment of this method of testing is the direct determination of thenucleotide sequence of the patient's OCTN2 gene. For example, using theabove oligonucleotide as the primer, the whole OCTN2 gene or a part ofit is amplified by the Polymerase Chain Reaction (PCR) using as thetemplate a DNA isolated from a patient suspected of having a diseasecaused by an OCTN2 mutation. By comparing this sequence with that of ahealthy individual, it is possible to conduct a test for a diseasearising from an OCTN2 gene mutation.

As the testing method of the invention, other than determining thenucleotide sequence of DNA derived directly from the patient, severalother methods are also used. One such embodiment comprises the followingsteps of: (a) preparing a DNA sample from a patient; (b) amplifying thepatient-derived DNA using the primer of this invention; (c) dissociatingamplified DNA into single-stranded DNA; (d) separating the dissociatedsingle-stranded DNA on a non-denaturing gel; and, (e) comparing themobility of separated single stranded DNA on the gel with a control froma healthy individual.

An example of such a method is the PCR-single-strand conformationpolymorphism (PCR-SSCP) method (Cloning and polymerase chainreaction-single-strand conformation polymorphism analysis of anonymousAlu repeats on chromosome 11, Genomics, 1992 Jan 1, 12(1):139-146;Detection of p53 gene mutations in human brain tumors by single-strandconformation polymorphism analysis of polymerase chain reactionproducts, Oncogene, 1991 August 1, 6(8):1313-1318; Multiplefluorescence-based PCR-SSCP analysis with postlabeling, PCR MethodsAppl. 1995 April 1, 4(5):275-282). This method is comparatively easy tohandle, and has various advantages such as requiring only a small amountof a sample, and therefore, is suitable for screening a large number ofDNA samples. The principle of this method is as follows. When a doublestranded DNA fragment is disassociated into single strands, each strandforms an original high-order structure depending on its nucleotidesequence. When these dissociated DNA strands are electrophoresed withina polyacrylamide gel free of denaturants, the single stranded DNAs thatare complementary and have the same length, migrate to differentpositions according to the difference in their high-order structure.This high order structure of the single strands change even by a singlenucleotide substitution showing different mobilities in polyacrylamidegel electrophoresis. Therefore, the presence of a mutation in a DNAfragment due to point mutation, deletion, or insertion can be detectedby the change in mobility.

Specifically, first, the whole OCTN2 gene or a part of it is amplifiedby PCR, and such. A length of 200 to 400 bp is usually preferredamplified range. Regions amplified include all the exons and all theintrons of the OCTN2 gene, as well as the promoter and enhancer of theOCTN2 gene. PCR can be done, for example, according to conditionsdescribed in Example 1. When amplifying the gene fragment by PCR, aprimer labeled by an isotope such as ³²p, a fluorescent dye, or biotinis used, or the DNA fragment synthesized by PCR after adding a substratenucleotide labeled by an isotope such as ³²p, a fluorescent dye, orbiotin, is labeled. Labeling can also be done by adding to thesynthesized DNA fragment a substrate nucleotide labeled by an isotopesuch as ³²p, a fluorescent dye, or biotin, using the Klenow enzyme andsuch after the PCR reaction. The labeled DNA fragment thus obtained isdenatured by heating and such, and electrophoresed in a polyacrylamidegel free of denaturants such as urea. Conditions for separating the DNAfragment can be improved by adding a suitable amount (about 5 to 10%) ofglycerol to the polyacrylamide gel. Conditions of electrophoresis varydepending on the properties of the DNA fragment, but room temperature(from 20 to 25° C.) is usually used. When a preferable separation cannotbe accomplished, the temperature that gives the optimum mobility at 4 to30° C. is evaluated. Following electrophoresis, the mobility of the DNAfragment is detected by an autoradiography using X-ray films, a scannerthat detects fluorescence, and so on, and analyzed. When a band having adifference in mobility is detected, this band is directly excised fromthe gel, re-amplified by PCR, and is directly sequenced to verify thepresence of a mutation. Even when labeled DNA is not used, the band canbe detected by staining the gel after electrophoresis with ethidiumbromide, silver, and such.

Another embodiment of the test method of the present invention comprisesthe following steps of: (a) preparing a DNA sample from a patient; (b)amplifying patient-derived DNA using the primer of this invention; (c)cleaving the amplified DNA;

(d) separating the DNA fragments according to their size; (e)hybridizing the probe DNA of the invention labeled with a detectablelabel to the DNA fragments separated; and(f) comparing the size of the detected DNA fragment with a control froma healthy individual.

Such methods include those using Restriction Fragment LengthPolymorphism (RFLP), PCR-RFLP method, and so on. Restriction enzymes areusually used to cleave DNA. Specifically, compared to a DNA fragment ofa healthy individual, the size of one obtained following restrictionenzyme treatment changes when a mutation exists at the recognition siteof the restriction enzyme, or when nucleotides have been inserted ordeleted in the DNA fragment resulting from restriction enzyme treatment.The portion containing the mutation is amplified by PCR, the amplifiedproducts are treated with each restriction enzyme and electrophoresed todetect the mutation as the difference of mobility. Alternatively,chromosomal DNA is cleaved with these restriction enzymes, and afterelectrophoresis, the presence or absence of a mutation can be detectedby southern-blotting using the probe DNA of the invention. Therestriction enzymes used can be suitably selected according to eachmutation. This method can use not only genomic DNA, but also cDNA madeby treating RNA prepared from patients with reverse transcriptase,cleaving this cDNA as-it-is with restriction enzymes, and thenconducting southern blotting. It is also possible to examine the changesin mobility after amplifying the whole OCTN2 gene, or a part of it, byPCR using the above cDNA as the template, and cleaving the amplifiedproducts by restriction enzymes.

A similar detection is also possible using RNA prepared from patientsinstead of DNA. This method includes the steps of: (a) preparing an RNAsample from a patient; (b) separating the prepared RNA according totheir size; (c) hybridizing the probe DNA of the invention labeled by adetectable label to the separated RNA; and (d) comparing the size of thedetected RNA with a control from a healthy individual. In a specificexample of this method, RNA prepared from a patient is electrophoresed,northern blotting is done using the probe of the invention to detect themobility change.

Another embodiment of the method of the invention comprises the stepsof:

(a) preparing a DNA sample from a patient; (b) amplifyingpatient-derived DNA using the primer of this invention; (c) separatingthe amplified DNA on a gel in which the concentration of the DNAdenaturant gradually increases; and, (d) comparing mobility of the DNAseparated upon the gel with a control from a healthy individual.

An example of such a method is denaturant gradient gel electrophoresis(DGGE). The whole OCTN2 gene or a part of it is amplified by a methodsuch as PCR using the primer of the invention, and the amplified productis electrophoresed in a gel in which the concentration of the DNAdenaturant gradually increases, and compared with a control from ahealthy individual. In the case of a DNA having a mutation, the DNAfragment will become single stranded at a low denaturant concentrationand the moving speed will become extremely slow. The presence or absenceof a mutation can be detected by detecting the change in mobility.

Allele Specific Oligonucleotide (ASO) hybridization can be usedalternatively when the aim is to detect a mutation at a specific site.When an oligonucleotide comprising a nucleotide sequence thought to havea mutation is prepared and this is hybridized with sample DNA, thehybrid formation efficiency will decrease when there is a mutation. Thiscan be detected by southern blotting and by a method using the propertyof special fluorescent reagents that quench when intercalated into ahybrid gap. The detection by ribonuclease A mismatch cleavage method canalso be used. Specifically, the whole OCTN2 gene, or a part of it, isamplified by a method such as PCR, and the amplified product ishybridized to labeled RNA prepared from OCTN2 cDNA and such incorporatedinto a plasmid vector, etc. The hybrids will be single stranded in theportion where a mutation exists. This portion is cleaved by ribonucleaseA and the existence of a mutation can be detected by autoradiography,and such.

The present invention also relates to a test drug for systemic carnitinedeficiency that comprises an antibody binding to the OCTN2 protein asthe active ingredient. An antibody binding to the OCTN2 protein can beprepared using methods well known to those skilled in the art.Polyclonal antibodies can be made by, obtaining the serum of smallanimals such as rabbits immunized with the OCTN2 protein (apart from thenatural protein, recombinant OCTN2 proteins expressed in suitable hostcells (E. coli, yeasts, mammals, and such), such as recombinant OCTN2protein expressed in E. coli as a fusion protein with GST) of thepresent invention, or a partial peptide. The serum is then purified by,for example, ammonium sulfate precipitation, protein A or protein Gcolumn chromatography, DEAE ion exchange chromatography, or an affinitychromatography using a column to which the protein of the presentinvention or synthetic peptide is coupled. Monoclonal antibodies can bemade by immunizing small animals such as mice with the OCTN2 protein ora partial peptide thereof, excising the spleen from the mouse,homogenizing it and separating cells, fusing the cells with mousemyeloma cells using a reagent such as polyethylene glycol, and selectingclones that produce an antibody binding to the OCTN2 protein from thefused cells (hybridomas) produced. Next, the obtained hybridomas aretransplanted into the abdominal cavity of a mouse, and ascites areextracted from the mouse. The obtained monoclonal antibodies can bepurified by, for example, ammonium sulfate precipitation, protein A orprotein G column chromatography, DEAE ion exchange chromatography, or anaffinity chromatography using a column to which the OCTN2 protein orsynthesized peptide is coupled. When using the antibody as a test drug,it is mixed with sterile water, physiological saline, plant oils,surfactants, lipids, solubilizers, stabilizers (BSA, gelatin, etc.),preservatives, and such, according to needs. An example of a test forsystemic carnitine deficiency features the staining of tissues collectedor cells isolated from a patient by the enzyme-labeled antibody method,fluorescence-labeled antibody method, and test for a deficiency,abnormal accumulation, or abnormal intracellular distribution of theOCTN2 protein. Testing can also be done by preparing a cell-extract oftissues collected or cells isolated from a systemic carnitine deficiencypatient, separating the cell-extract by methods such as SDS-PAGE,transferring onto a nitrocellulose membrane, PVDF membrane, and such,and then staining this by a method (western blotting, immunoblotting,etc) using the above-described enzyme-labeled antibody method, etc.

The present invention also relates to a therapeutic drug for systemiccarnitine deficiency. One such embodiment has the OCTN2 gene as theactive ingredient. When using the OCTN2 gene as a therapeutic drug, itis given to the patient by oral, intravenous, topical administration andsuch, as the full length OCTN2 chromosomal DNA, a part of it, or byincorporating the OCTN2 DNA into a suitable vector, for example,adenovirus vector, adeno associated virus vector, retro virus vector, orplasmid DNA. The ex vivo method can also be used for administrationapart from the in vivo method. The transition and absorption intotissues can be enhanced by enclosing the gene in a liposome prepared bymicellization of phospholipids, or by adding a cationic lipid andforming a complex with genomic DNA. Therefore, the method of theinvention can replace a patient's mutated OCTN2 gene by a normal gene,and also additionally administer the normal gene, thereby enabling thetreatment of systemic carnitine deficiency.

Another embodiment of the invention relating to a therapeutic drug ofsystemic carnitine deficiency comprises the OCTN2 protein as the activeingredient. The amino acid sequences of human and mouse OCTN2 proteinsare shown in SEQ ID NOs: 1 and 3, respectively. The OCTN2 protein can beprepared as a natural protein and also as a recombinant protein. Thenatural protein can be prepared by a method well known to one skilled inthe art, for example, by isolating the OCTN2 protein from tissues orcells that show a high level expression of the protein (e.g. fetalkidney) by affinity chromatography using an antibody against a partialpeptide of the OCTN2 protein. On the other hand, a recombinant proteincan be prepared by culturing cells transformed by DNA (for example, SEQID NO:2) encoding the OCTN2 protein. Cells used for the production ofrecombinant proteins include mammalian cells such as, COS cells, CHOcells, and NIH3T3 cells, insect cells such as sf9 cells, yeast cells,and E. coli cells. Vectors for expressing the recombinant proteinswithin cells vary according to the host used, and normally, pcDNA3(Invitrogen), pEF-BOS (Nucleic Acids Res. 1990, 18(17), 5322) and suchare used as vectors for mammalian cells, the “BAC-to-BAC baculovirusexpression system” (GIBCO BRL) and such are used for insect cells,“Pichia Expression Kit” (Invitrogen) and such are used for yeast cells,pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen) and such are usedfor E. coli cells. Vectors are introduced to hosts using, for example,the calcium phosphate method, DEAE dextran method, method using cationicliposome DOTAP (Boehringer Mannheim), and Superfect (Qiagen),electroporation method, calcium chloride method, and such. Therecombinant protein can be purified from the transformant obtainedusually using methods described in “The Qiaexpressionist handbook,Qiagen, Hilden, Germany”.

When using the obtained OCTN2 protein as a therapeutic drug for treatingsystemic carnitine deficiency, the OCTN2 protein can be directlyadministered, or can be given after being formulated into apharmaceutical composition by a well-known pharmaceutical manufacturingmethod. For example, the drug can be given after suitably combining witha generally used carrier or medium such as, sterilized water,physiological saline, plant oils, surfactants, lipids, solubilizers,stabilizers, preservatives, and such.

The dosage varies depending on factors such as the patient's bodyweight, age, healthiness, and method of administration, but a skilledartisan can suitably select the dosage. Usually, it is within the rangefrom 0.01 to 1000 mg/kg. The administration can be done orally,intravenously, intramuscularly, or percutaneously. A skilled artisan caneasily replace, add, or delete amino acid(s) in the amino acid sequenceof the OCTN2 protein using a well-known method such as the site-specificmutation induction system using PCR (GIBCO-BRL, Gaithersburg, Maryland),site-specific mutagenesis using oligonucleotides (Kramer, W. and Fritz,H J, 1987, Methods in Enzymol, 154:350-367), the Kunkel method (MethodsEnzymol., 1988, 85:2763-2766), and such.

Another embodiment of the therapeutic drug for systemic carnitinedeficiency uses a compound that enhances the activity of the OCTN2protein as the active ingredient. Such a compound can be screened asfollows. For example, a plasmid expressing the OCTN2 protein isconstructed, and this is introduced into HEK293 cells by the calciumphosphate method. Radiolabeled carnitine and a test compound are addedto this transformant and the carnitine transporting activity into thecells is determined. A compound that can enhance the carnitinetransporting activity is selected by comparing with the activity of theOCTN2 protein in the absence of the test compound. See Japanese PatentApplication Hei 9-260972 and Hei 10-156660 for the detailed method.

Similar to the above-mentioned use of the OCTN2 protein as a therapeuticdrug, the isolated compound can also be formulated into a pharmaceuticalcomposition using well-known pharmaceutical manufacturing methods. Thedose range is usually within 0.01 to 1000 mg/kg.

It is also conceivable to utilize the region regulating OCTN2 geneexpression or a factor that binds to this region for the treatment ofsystemic carnitine deficiency.

The OCTN2 gene comprising the region that regulates OCTN2 geneexpression is useful in the above-mentioned gene therapy as it canexpress the OCTN2 gene under normal expression regulation in vivo byintroducing it into patients who lack the OCTN2 gene, or who have adefect in OCTN2 gene expression.

Moreover, if the promoter site is determined from the upstream region ofthe OCTN2 gene, a compound that regulates OCTN2 gene expression amountcan be simply screened by using a reporter gene expression vector havingthe above promoter site through examining the influence of variouscompounds on the production of reporter gene products. Such a screeningmethod comprises the following steps of, (a) constructing a vector inwhich a reporter gene is ligated to the downstream of the promoter site,(b) introducing the vector into a suitable cell, and, (c) detecting thereporter gene activity by contacting or introducing a test compound tothe above cell. Examples of the test compound include, proteins,peptides, synthetic compounds, natural compounds, genes, gene products,and such.

A compound regulating OCTN2 gene expression can also be screened bycontacting a test sample with the promoter site, and selecting acompound (such as a protein) that binds to the promoter site. Forexample, a synthetic oligo DNA and such having the nucleotide sequenceof the promoter site is prepared, this is bound to a suitable supportsuch as Sepharose, and contacted with a cell-extract, and such. Then, atranscription factor and such that binds to this promoter site andregulates OCTN2 gene expression can be purified by, for example,affinity chromatography.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the direct sequencing of the mouse OCTN2 gene amplified byRT-PCR. wt/wt shows wild-type homologous mouse (SEQ ID NO:27), andjvs/jvs shows the jvs homologous mouse (SEQ ID NO:28). OCTN2 gene of thejvs mouse has a mutation at the nucleotide shown by the arrow.

FIG. 2 is electrophoretic images showing the mutation in the OCTN2 geneof the jvs mouse, which was detected using the PCR-RFLP method (Cfr 131cleavage). The fragment shown by the arrow head derives from the normalgene, and the fragments shown by the arrows were due to the mutatedgene.

FIG. 3 shows results of the carnitine transporting activity assay ofwild-type mouse OCTN2 and the mutant mouse OCTN2. A sodium-dependentcarnitine transporting activity is seen for the wild type, whereas themutant (Jvs) shows absolutely no activity. “Mock” is when acDNA-non-containing vector was used as the control.

FIG. 4 is an electrophoretic image showing the results of western blotanalysis using anti myc antibody. It can be seen that the wild-typeOCTN2 protein (wild) and the mutant OCTN2 protein (Jvs) is produced insimilar amounts. “Mock” is when a cDNA-non-containing vector was used asthe control.

FIG. 5 shows the results of OCTN2 gene analysis in the KR family. Thepedigree chart of this family is shown on top. Squares indicate males,circles females, filled ones individuals having systemic carnitinedeficiency, and crossed squares indicate deceased individuals. Anelectrophoretic image showing the PCR results is given below. “N” showsthe results of the normal gene used as the control. The fragments shownby the arrowhead are PCR products derived from the normal gene, and thefragments shown by the arrow derived from the gene where the defectexits.

FIG. 6 shows the results of sequencing exon 1 of the OCTN2 gene.Compared to the normal OCTN2 gene (upper panel; wild-type; nucleotides214 to 234 of SEQ ID NO:5), the OCTN2 of systemic carnitine deficiencypatients (lower panel; SEQ ID NO:29) belonging to the AK family, show aninsertion of a cytosine residue at the position indicated by the arrow.

FIG. 7 shows the results of sequencing exon 2 of the OCTN2 gene.Compared to the normal OCTN2 gene (upper panel; wild-type; nucleotides8,630 to 8,648 of SEQ ID NO:5), the OCTN2 of systemic carnitinedeficiency patients (lower panel; SEQ ID NO:30) belonging to the AKfamily, show a single nucleotide substitution (A has substituted G) asindicated by the arrow.

FIG. 8 is electrophoretic images showing the results of the analysis oftwo-types of mutations seen in the OCTN2 gene of a systemic carnitinedeficiency patient belonging to the AK family using a PCR-RFLP methodutilizing BcnI and NlaIV, respectively. The pedigree chart of thisfamily is shown on top. Square indicates a male, circles females, andthe filled circle indicates a systemic carnitine deficiency patient. “N”shows the results of the normal gene used as the control. The fragmentsshown by the arrows derived from the mutant gene.

FIG. 9 shows the results of the sequencing analysis of the intron 8/exon9 of the OCTN2 gene. Compared to the normal gene (normal; nucleotides23,925 to 23,943 of SEQ ID NO:5), the gene deriving from the patientbelonging to the TH family (patient; SEQ ID NO:31) has a splicing sitemutation (AG to AA) in the 3′ end of intron 8. The pedigree chart ofthis family is shown on top. Squares indicate males, the circle afemale, and filled square indicates a systemic carnitine deficiencypatient.

DETAILED DESCRIPTION OF THE INVENTION

The invention shall be described in detail below, but it is not to beconstrued as being limited thereto.

Example 1

Proof in mouse and human showing that the gene responsible for systemiccarnitine deficiency (SCD) is OCTN2

The Inventors have previously isolated human cDNA encoding a proteinhaving an activity to transport carnitine in a sodium-ion dependentmanner, and also the corresponding mouse cDNA (Japanese PatentApplication No. Hei 9-260972, Japanese Patent Application No. Hei10-156660). The nucleotide sequences of the human and mouse OCTN2 cDNAisolated by the Inventors are shown in SEQ ID NO:2 and 4, respectively,and the amino acid sequences of the proteins encoded by these cDNAs areshown in SEQ ID NO: 1 and 3, respectively.

The Inventors drew up a working hypothesis that OCTN2 might be the generesponsible for systemic carnitine deficiency, and conducted experimentsto prove this.

(1) OCTN2 Gene Analysis in Juvenile Visceral Steatosis (jvs) Mouse

The juvenile visceral steatosis (jvs) mouse was generated due to amutation in the C3H.OH mouse. This jvs mouse shows symptoms similar tosystemic carnitine deficiency patients, and shows an extremely lowcarnitine concentration within its blood and tissues. This phenotype isinherited by autosomal inheritance. From the above facts, the jvs mouseis considered to be a mouse model for systemic carnitine deficiency(Hashimoto, N. et al., Gene-dose effect on carnitine transport activityin embryonic fibroblasts of JVS mice as a model of human carnitinetransporter deficiency, Biochem Pharmacol, 1998, 55:1729-1732). TheInventors examined the OCTN2 gene arrangement of the jvs mouse.Specifically, whole RNA was extracted from the kidney of a jvshomologous mouse, cDNA was synthesized, jvs mouse OCTN2 cDNA wasamplified using this synthesized cDNA as the template by RT-PCR, and thesequence was examined by direct sequencing.

The amplification reaction by PCR was conducted as follows. For the 5′side fragment, the primers MONB 31(5′-gataagcttacggtgtccccttattcccatacg-3′/SEQ ID NO:22) and MONB 20(5′-cccatgccaacaaggacaaaaagc-3′/SEQ ID NO:23) were prepared. Then,amplification was done within a reaction solution (50 μl) containing,cDNA, 5 μl of 10× KOD buffer (Toyobo), 5 μl of 2 mM dNTPs, 2 μl of 25 mMMgCl₂, 0.5 μl of KOD DNA polymerase (Toyobo), 1 μl of 20 μM MONB 31primer, and 1 μl of 20 μM MONB 20 primer at 94° C. for 3 min, 30 cyclesof “94° C. for 30 sec, 50° C. for 30 sec, and 74° C. for 1 min”, and 72°C. for 10 min. As for the 3′ side fragment, the primers MONB 6(5′-tgtttttcgtgggtgtgctgatgg-3′/SEQ ID NO:24) and MONB 26(5′-acagaacagaaaagccctcagtca-3′/SEQ ID NO:25) were prepared, andamplification was done within a reaction solution (50 μl) containingcDNA, 5 μl of 10× ExTaq buffer (TaKaRa), 4 μl of 2.5 mM dNTPs, 1 μl of amixture of ExTaq DNA polymerase (TaKaRa) and anti Taq antibody (TaqStartantibody™, CLONTECH), 1 μl of 20 μM MONB 6 primer, and 1 μl of 20 μMMONB 26 primer, at 94° C. for 2 min, 30 cycles of “94° C. for 30 sec,60° C. for 30 sec, and 74° C. for 2 min”, and 72° C. for 10 min.

Sequencing revealed that the codon encoding the 352^(nd) leucine (CTG)was mutated to a codon encoding arginine (CGG) (FIG. 1). This mutationcan be detected by Restriction Fragment Length Polymorphism (PCR-RFLP)due to the presence of the Cfr13I restriction enzyme site. This methodrevealed that the jvs homologous mouse (jvs/jvs) had this mutation inboth alleles, and that the heterologous mouse (wt/jvs) has both themutated and wild type alleles (FIG. 2 left). This mutation was alsofound in the C57BL jvs mouse in which the genetic background has beenreplaced with that of the C57BL/6 mouse by backcrossing 12 times or more(FIG. 2 right). Since the C57BL jvs mouse was constructed after a seriesof selections using the jvs phenotype as an index, the jvs phenotype andOCTN2 mutations are considered to be very closely associated.

Next, the effect this mutation has on the carnitine transportingactivity was examined. Plasmid DNA expressing wild-type mouse OCTN2, andthose expressing mutated OCTN2 were separately introduced into BEK293cells, and then, carnitine transporting ability was measured similar tothe assay of human OCTN2 described in Japanese Patent Application Hei10-156660 (FIG. 3). This revealed that although wild-type mouse OCTN2shows a carnitine transporting activity similar to human OCTN2, themutated OCTN2 has absolutely no activity. However, both proteins wereconfirmed to be expressed at a similar amount by a western blottingusing an antibody against the c-myc epitope sequence(NH2-EQKLISEEDL-COOH; SEQ ID NO:26) added to the C terminus (FIG. 4).

Thus, the jvs mouse is thought to have developed the disease due to afunctional deletion mutation of the OCTN2 gene.

(2) OCTN2 gene analysis in human systemic carnitine deficiency patients

A database search using human OCTN2 cDNA sequence revealed that thehuman OCTN2 genomic DNA sequence has been decoded by Lawrence BerkeleyNational Laboratory (LBNL) of the United States as a part of the humangenome project. However, it was only recorded as several cosmid clonesequences, therefore, the inventors determined a complete human OCTN2genomic DNA sequence (SEQ ID NO:5) by comparing with human OCTN2 cDNAsequence and suitably combining the clone sequences. The human OCTN2gene is an about 26 kb gene comprising ten exons and nine introns. Theeight pairs of primers shown below, which can amplify all the exons aseight fragments, were prepared from this gene arrangement.

Specifically, OCN2 43 (5′-GCAGGACCAAGGCGGCGGTGTCAG-3′, SEQ ID NO:6) andOCN2 44 (5′-AGACTAGAGGAAAAACGGGATAGC-3′, SEQ ID NO:7) for exon one; OCN225 (5′-AGATTTTTAGGAGCAAGCGTTAGA-3′ SEQ ID NO:8) and OCN2 26(5′-GAGGCAGACACCGTGGCACTACTA-3′, SEQ ID NO:9) for exon two; OCN2 27(5′-TTCACACCCACTTACTGGATGGAT-3′ SEQ ID NO: 10) and OCN2 50(5′-ATTCTGTTTTGTTTTGGCTCTTTT-3′, SEQ ID NO: 11) for exons three andfour; OCN2 31 (5′-AGCAGGGCCTGGGCTGACATAGAC-3′, SEQ ID NO: 12) and OCN232 (5′-AAAGGACCTGACTCCAAGATGATA-3′, SEQ ID NO: 13) for exon five; OCN233 (5′-TCTGACCACCTCTTCTTCCCATAC-3′, SEQ ID NO: 14) and OCN2 34(5′-GCCTCCTCAGCCACTGTCGGTAAC-3′, SEQ ID NO: 15) for exon six; OCN2 35(5′-ATGTTGTTCCTTTTGTTATCTTAT-3′, SEQ ID NO: 16) and OCN2 36(5′-CTTGTTTTCTTGTGTATCGTTATC-3′, SEQ ID NO:17) for exon seven; OCN2 37(5′-TATGTTTGTTTTGCTCTCAATAGC-3′, SEQ ID NO:18) and OCN2 40(5′-TCTGTGAGAGGGAGTTTGCGAGTA-3′, SEQ ID NO: 19) for exon eight and nine;and, OCN2 41 (5′-TACGACCGCTTCCTGCCCTACATT-3′, SEQ ID NO:20) and OCN2 42(5′-TCATTCTGCTCCATCTTCATTACC-3′, SEQ ID NO:21) for exon 10.

Next, human OCTN2 gene mutations in three families that have systemiccarnitine deficiency patients, but no blood relationships were examined.The analysis is done by amplifying all the exons using the above primersand genomic DNA prepared from blood cells as the template, andsubjecting the amplified products into direct sequencing.

The amplification reaction by PCR was done within a reaction solution(50 μl) containing 100 ng of genomic DNA, 5 μl of 10× ExTaq buffer(TaKaRa), 4 μl of 2.5 mM dNTPs, 1 μl of a mixture of ExTaq DNApolymerase (TaKaRa) and anti Taq antibody (TaqStart antibody™,CLONTECH), and 1 μl of each of the 20 μM primers. The reactionconditions were, 94° C. for 2 min, 36 cycles of “94° C. for 30 sec, 60°C. for 30 sec, and 74° C. for 2 min”, and 72° C. for 10 min. However, inthe case of exon one and exon five amplification, a reaction solution(50 μl) containing 100 ng genomic DNA, 25 μl of 2× GC buffer 1 (TaKaRa),8 μl of 2.5 mM dNTPs, 0.5 μl of LA Taq DNA polymerase (TaKaRa), and 1 μlof each of the 20 μM primers, was used.

In the first family (KR family), a 113 bp deletion was found in firstexon of the OCTN2 gene of a systemic carnitine deficiency patient (FIG.5). This deletion affects the initiation codon and thus, a completeprotein will not be produced. The next usable ATG codon present in thecorrect frame is at nucleotide no. 177, and in this case, it is thoughtthat at least two transmembrane regions will be deleted. The twosystemic carnitine deficiency patients in this family were found tocontain this mutated OCTN2 gene in both alleles. On the other hand, theparents and the two brothers of the patient, who have not developed thedisease, carry the mutation on just one allele.

In the second family (AK family), the systemic carnitine patients werefound to contain two types of mutated OCTN2 genes. One mutation was acytosine insertion just after the initiation codon, which is thought tocause a frame shift and prevent the proper protein from being produced(FIG. 6). The other mutation is a single base substitution (G to A) inthe codon encoding the 132^(nd) tryptophan (TGG). This mutation hadaltered the codon into a stop codon (TGA) (FIG. 7). These mutations arethought to prevent the production of active OCTN2 proteins in patients.These mutations can be detected by PCR-RFLP analysis using BcnI, NlaIVrestriction enzymes, respectively, which revealed that the patient'sparents who have not developed the disease, had one of each of themutations, and the patient's sisters who have not developed the disease,do not have any mutated genes (FIG. 8).

In the third family (TH family), a mutation (AG to AA) was found in thesplicing site in the 3′ end of the intron eight of the OCTN2 gene (FIG.9). This mutation prevents the gene from undergoing normal splicing, andthus, it is expected that the normal protein would not be produced.Sequencing analysis showed that the systemic carnitine deficiencypatient belonging to this family had this mutation in both alleles. Onthe other hand, the patient's parents and one of the brothers who havenot developed the disease had one mutated allele.

The above results revealed that systemic carnitine deficiency is agenetic disease caused by mutations in the OCTN2 gene. Thus, the presentinvention enables definitive diagnosis, prenatal diagnosis and such, ofsystemic carnitine deficiency by examining mutations in the OCTN2 geneusing analyses described herein, as well as other methods. The presentinvention also enables treatment of systemic carnitine deficiency bytreatments such as gene therapy using the OCTN2 gene.

INDUSTRIAL APPLICABILITY

The present invention revealed that the OCTN2 gene is the generesponsible for systemic carnitine deficiency, thus enabling tests forthe disease by detecting mutations in the OCTN2 gene and its protein.Moreover, the present invention facilitates treatment of systemiccarnitine deficiency by utilizing the OCTN2 gene and its protein.

1. A method of screening for a compound that regulates OCTN2 geneexpression, the method comprising: (a) contacting a test compound with(i) OCTN2 DNA encoding OCTN2; or (ii) a cell comprising said DNA; and(b) measuring OCTN2 gene expression, wherein an increase or decrease ingene expression in the presence of the test compound, compared to in theabsence of the test compound, is indicative of a compound that regulatesOCTN2 gene expression.
 2. The method of claim 1, wherein the OCTN2 DNAcomprises a promoter region or an enhancer region of the OCTN2 gene. 3.The method of claim 1, wherein the OCTN2 DNA comprises the nucleotidesequence of SEQ ID NO:5.
 4. The method of claim 1, wherein the testcompound is selected from among proteins, peptides, synthetic compounds,natural compounds, genes and gene products.
 5. The method of claim 4,wherein the test compound is a protein.
 6. The method of claim 5,wherein the protein is a transcription factor.
 7. A method of screeningfor a compound that regulates OCTN2 gene expression, the methodcomprising: (a) providing a vector comprising a reporter gene downstreamof a promoter sequence from SEQ ID NO:5; (b) contacting a test compoundwith the vector; and (c) detecting expression of the reporter gene,wherein an increase or decrease in expression of the reporter gene inthe presence of the test compound, compared to in the absence of thetest compound, is indicative of a compound that regulates OCTN2 geneexpression.
 8. The method of claim 7, wherein the test compound binds tothe promoter.
 9. A method of screening for a compound that regulatesOCTN2 gene expression, the method comprising: (a) providing anoligonucleotide DNA, wherein the oligonucleotide DNA comprises aregulatory sequence from SEQ ID NO:5 that is upstream of the OCTN2coding sequence; (b) contacting a test compound with the oligonucleotideDNA; and (c) determining whether the test compound binds to the oligoDNA, wherein binding to the oligonucleotide DNA is an indication thatthe test compound is potentially able to regulate OCTN2 gene expression.10. The method of claim 9, wherein the regulatory sequence is a promoterregion of the OCTN2 gene.
 11. A method of screening for a compound thatregulates OCTN2 gene expression, the method comprising: (a) contacting atest compound with DNA that comprises a promoter region of the OCTN2gene; and (b) measuring binding of the test compound to the promoter,wherein a compound that binds to the promoter is indicative of acompound that regulates OCTN2 gene expression.
 12. The method of claim11, wherein the DNA further comprises (i) OCTN2 genomic DNA (SEQ IDNO:5); (ii) a sequence that regulates transcription of SEQ ID NO:5; or(iii) DNA that encodes an OCTN2 protein comprising the amino acidsequence of SEQ ID NO:1.
 13. The method of claim 11, wherein the DNA isbound to a solid support.
 14. The method of claim 13, wherein thecompound is purified by affinity chromatography.
 15. The method of claim11, wherein the test compound is a protein.
 16. The method of claim 15,wherein the protein is a transcription factor.
 17. A method of screeningfor a compound that regulates OCTN2 gene expression, the methodcomprising: (a) providing a cell comprising a reporter gene downstreamof DNA that comprises a promoter region of the OCTN2 gene; and (b)detecting reporter gene activity in the cell, wherein an increase ordecrease in reporter gene expression in the presence of the testcompound, compared to in the absence of the test compound, is indicativeof a compound that regulates OCTN2 gene expression.
 18. The method ofclaim 17, wherein the test compound is a protein.
 19. The method ofclaim 18, wherein the protein is a transcription factor.
 20. A method ofscreening for a compound that enhances the activity of the OCTN2protein, the method comprising: (a) contacting a test compound with anOCTN2 protein comprising the amino acid sequence of SEQ ID NO: 1; and(b) evaluating the activity of the protein, wherein an increase inactivity in the presence of the test compound, compared to in theabsence of the test compound, is indicative of a compound that enhancesthe activity of the OCTN2 protein.
 21. The method of claim 20, whereinthe test compound is introduced into a cell comprising a vector thatexpresses the OCTN2 protein.
 22. The method of claim 21, wherein theactivity of the OCTN2 protein is evaluated by measuring carnitinetransport into the cell.
 23. A pharmaceutical composition comprising anisolated protein comprising the amino acid sequence of SEQ ID NO: 1 asan active ingredient, wherein the protein is in an amount effective toincrease cellular uptake of carnitine.
 24. A method of testing whetheran individual's genome carries a mutant OCTN2 allele that, in thehomozygous state, may result in systemic carnitine deficiency, themethod comprising (a) identifying an individual suspected of carryingthe mutant allele; and (b) analyzing a nucleic acid sample from theindividual to determine the presence or absence of a mutation in (i) DNAencoding OCTN2 (SEQ ID NO: 1) or (ii) OCTN2 genomic DNA (SEQ ID NO:5) or(iii) a sequence that regulates expression of SEQ ID NO:5, wherein thepresence of the mutation in (i) or (ii) or (iii) indicates that theindividual carries a mutant OCTN2 allele that, in the homozygous state,may result in systemic carnitine deficiency.